Support material for solid phase organic synthesis

ABSTRACT

A support material for solid phase synthesis is provided having an amine-containing organic group attached to it through a linker. The support material is of the following general formula (Formula I):

This is a division of application Ser. No. 08/665,509, filed Jun. 18,1996, now U.S. Pat. No. 5,917,015, which is incorporated herein byreference.

STATEMENT OF GOVERNMENT RIGHTS

The present invention was made with support from National Institutes ofHealth Grant No. GM 42722. The government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

Solid-phase peptide synthesis (SPPS) involves a covalent attachment step(i.e., anchoring) that links the nascent peptide chain to an insolublepolymeric support (i.e., support material) containing appropriatefunctional groups for attachment. Subsequently, the anchored peptide isextended by a series of addition (deprotection/coupling) cycles thatinvolve adding N^(α)-protected and side-chain-protected amino acidsstepwise in the C to N direction. Once chain assembly has beenaccomplished, protecting groups are removed and the peptide is cleavedfrom the support.

Typically, SPPS begins by using a handle to attach the initial aminoacid residue to the functionalized polymeric support. A handle (i.e.,linker) is a bifunctional spacer that, on one end, incorporates featuresof a smoothly cleavable protecting group, and on the other end, afunctional group, often a carboxyl group, that can be activated to allowcoupling to the functionalized polymeric support. Known handles includeacid-labile p-alkoxybenzyl (PAB) handles, photolabile o-nitrobenzylester handles, and handles such as those described by Albericio et al.,J. Org. Chem., 55, 3730-3743 (1990) and references cited therein, and inU.S. Pat. No. 5,117,009 (Barany) and U.S. Pat. No. 5,196,566 (Barany etal.).

For example, if the support material is prepared withamino-functionalized monomers, typically, the appropriate handles arecoupled quantitatively in a single step onto the amino-functionalizedsupports to provide a general starting point of well-defined structuresfor peptide chain assembly. The handle protecting group is removed andthe C-terminal residue of the N^(α)-protected first amino acid iscoupled quantitatively to the handle. Once the handle is coupled to thesolid-phase and the initial amino acid or peptide is attached to thehandle, the general synthesis cycle proceeds. The synthesis cyclegenerally consists of deprotection of the N^(α)-amino group of the aminoacid or peptide on the resin, washing, and, if necessary, aneutralization step, followed by reaction with a carboxyl-activated formof the next N^(α)-protected amino acid. The cycle is repeated to formthe peptide or protein of interest. Solid-phase peptide synthesismethods using functionalized insoluble supports are well known. See, forexample, Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); Barany andMerrifield, In The Peptides, Vol. 2, pp. 1-284 (1979); Gross, E. andMeienhofer, J., Eds., Academic: New York; and Barany et al., Int. J.Peptide Protein Res., 30, 705-739 (1987).

Most current methods of SPPS rely on the a-carboxyl function of theeventual C-terminal amino acid residue to achieve anchoring to thesupport. However, this approach limits SPPS to the formation of peptideshaving acid, amide, or monosubstituted amide functionality, for example,as the C-terminal functionality, unless more complex procedures areused. Furthermore, certain functionalities, such as aldehydes, cannottypically be obtained using this approach. Cyclic peptides are also notpossible using this method. Also, racemization of sensitive amino acidresidues in the synthesis of peptide acids is a problem using thismethod.

Side-chain anchoring, i.e., methods of SPPS that use amino acids withside-chain functional groups for attachment of peptides, is potentiallyuseful for the formation of unusual C-terminal functionalities as wellas cyclic peptides. However, side-chain anchoring is inherently limitedto certain trifunctional amino acids. Therefore, it would be desirableto develop a general method of SPPS that: (1) allows for the preparationof a wider variety of peptides; (2) does not typically result inracemization of sensitive amino acid residues; and (3) can incorporate awider variety of amino acids into cyclic peptides.

SUMMARY OF THE INVENTION

The present invention provides a method of forming a support materiallinked to an amine-containing organic group for solid phase organicsynthesis comprising:

(a) attaching a preformed divalent linker to a support material; and

(b) attaching an amine-containing organic group to the preformeddivalent linker;

wherein steps (a) and (b) are carried out to form a support materiallinked to an amine-containing organic group of the following formula(Formula I):

 wherein:

(i) Ŝ represents a support material;

(ii) L represents a divalent linker;

(iii) Y represents H or a protecting group; and

(iv) R¹, R², and R³ are each independently H or an organic group.

A preferred divalent linker (L) is of the formula (Formula II):

wherein:

each U is independently selected from the group consisting of an alkylgroup, an alkoxy group, an aryl group, an alkoxyaryl group, an aralkylgroup, an aralkoxy group, an alkylthio group, an arylthio group, analkylamido group, an alkylsulfinyl group, an alkylsulfonyl group, analkylsulfoxide group, a halogeno group, and a nitro group, wherein anytwo U groups can be joined to form a ring; W is a functionalized spacergroup for anchoring the linker to the support material; R⁵ and R⁶ areeach independently H, an alkyl group, or an aryl group; and x=0-4.

To synthesize an organic compound, such as a peptide, once the supportmaterial of Formula I is prepared, a second organic group is attached tothe N atom to build an organic compound. This is done using standardsolid phase synthesis techniques and repeated addition cycles ofdeprotection and coupling.

The present invention also provides a method of synthesizing an organiccompound comprising:

(a) providing an aldehyde-functionalized support material having thefollowing formula (Formula III):

 wherein:

(i) Ŝ represents a support material;

(ii) V is NH, S or O;

(iii) T is O, NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group, anarylene group, or an aralkylene group;

(iv) R⁷ is an alkylene group, an arylene group, or an oxyalkylene group;

(v) each U is independently selected from the group consisting of analkyl group, an alkoxy group, an aryl group, an alkoxyaryl group, anaralkyl group, an aralkoxy group, an alkylthio group, an arylthio group,an alkylamido group, an alkylsulfinyl group, an alkylsulfonyl group, analkylsulfoxide group, a halogeno group, and a nitro group, wherein anytwo U groups can be joined to form a ring;

(vi) x=0-4; and

(vii) n=1-18;

(b) attaching an amine-containing organic group to the aldehydefunctionality under reducing conditions; and

(c) attaching a second organic group to the N atom of theamine-containing organic group to build an organic compound.

The aldehyde-functionalized support material of Formula III is alsoprovided, along with a kit for synthesizing an organic compound. The kitincludes an aldehyde-functionalized support material having the FormulaIII and instructions for preparing an organic compound on thealdehyde-functionalized support material.

The present invention also provides a support material linked to anamine-containing organic group for solid-phase synthesis of an organiccompound, wherein the support material has the following formula(Formula I):

wherein:

(a) Ŝ represents a support material;

(b) L represents a divalent linker;

(c) Y represents H or a protecting group;

(d) R¹ and R² are each independently H or an organic group; and

(e) R³ is an organic group having a protecting group Z that is removableunder mild conditions.

The present invention also provides a preformed linker having anamine-containing organic group attached thereto, of the formula (FormulaIV):

wherein:

(a) L represents a divalent linker;

(b) Q represents a group selected from the group consisting of C(O)OH,C(O)OPfp, C(O)F, C(O)Br, C(O)Cl, OH, Br, Cl;

(c) Y represents H or a protecting group;

(d) R¹ and R² are each independently H or an organic group; and

(e) R³ is an organic group having a protecting group Z that is removableunder mild conditions.

As used herein, the term “organic group” means a hydrocarbon group thatis classified as an aliphatic group, cyclic group, or combination ofaliphatic and cyclic groups (e.g., aralkyl groups). In the context ofthe present invention, the term “aliphatic group” means a saturated orunsaturated linear or branched hydrocarbon group. This term is used toencompass alkyl, alkenyl, and alkynyl groups, for example. The term“alkyl group” means a saturated linear or branched hydrocarbon groupincluding, for example, methyl, ethyl, isopropyl, t-butyl, heptyl,dodecyl, octadecyl, amyl, 2-ethylhexyl, and the like. The term “alkenylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon double bonds, such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched hydrocarbongroup with one or more carbon-carbon triple bonds. The term “cyclicgroup” means a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polycyclic aromatic hydrocarbon group. The term“heterocyclic group” means a closed ring hydrocarbon in which one ormore of the atoms in the ring is an element other than carbon (e.g.,nitrogen, oxygen, sulfur, etc.).

As is well understood in this technical area, a large degree ofsubstitution is not only tolerated, but is often advisable. Substitutionis anticipated on the materials of the present invention. As a means ofsimplifying the discussion and recitation of certain terminology usedthroughout this application, the terms “group” and “moiety” are used todifferentiate between chemical species that allow for substitution orthat may be substituted and those that do not allow or may not be sosubstituted. Thus, when the term “group” is used to describe a chemicalsubstituent, the described chemical material includes the unsubstitutedgroup and that group with O, N, or S atoms, for example, in the chain aswell as carbonyl groups or other conventional substitution. Where theterm “moiety” is used to describe a chemical compound or substituent,only an unsubstituted chemical material is intended to be included. Forexample, the phrase “alkyl group” is intended to include not only pureopen chain saturated hydrocarbon alkyl substituents, such as methyl,ethyl, propyl, t-butyl, and the like, but also alkyl substituentsbearing further substituents known in the art, such as hydroxy, alkoxy,alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus,“alkyl group” includes ether groups, haloalkyls, nitroalkyls,carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc. On the other hand, thephrase “alkyl moiety” is limited to the inclusion of only pure openchain saturated hydrocarbon alkyl substituents, such as methyl, ethyl,propyl, t-butyl, and the like.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for preparing organic compounds,particularly peptides, using solid-phase synthesis. The method involvesanchoring an amine-containing organic group, which is the starting pointfor building the organic compound, to a support material using adivalent linker. Specifically, if peptides are being prepared, the aminoacid residue or peptide is anchored through its backbone to the supportmaterial. This is in contrast to conventional methods of solid-phasepeptide synthesis, for example, that involve anchoring theamine-containing organic group through a side-chain functional group ofan amino acid residue, or through the a-carboxyl functionality of theeventual C-terminal amino acid residue.

This backbone amide linker (BAL) approach for the preparation ofpeptides avoids some of the aforementioned problems and allows for thepreparation of peptides or other organic compounds having a variety ofC-terminal functionalities, e.g., not only acids, but also thioacids andthioesters, alcohols, disubstituted amides, and aldehydes, among others.It also allows for the preparation of cyclic peptides using a widervariety of amino acids. Also, this approach is advantageous becausethere is little racemization of sensitive amino acid residues at roomtemperature.

The support material linked to an amine-containing organic group has thefollowing general formula (Formula I):

wherein:

Ŝ represents a support material, typically a solid support, which mayinclude a variety of functional groups for attachment, and may or maynot include a spacer; L represents a divalent linker; Y represents H ora protecting group, such as an N^(α)-amine protecting group; and R¹, R²,and R³ are each independently H or an organic group.

The support material of Formula I is prepared by attaching a preformeddivalent linker to a support material, and attaching an amine-containingorganic group to the preformed divalent linker. These two steps can becarried out in either order. That is, the amine-containing organic groupcan be attached to the preformed divalent linker prior to attaching thepreformed divalent linker to the support material. Alternatively, thepreformed divalent linker can be attached to the support material priorto attaching the amine-containing organic group to the preformeddivalent linker.

As used herein, a preformed divalent linker (i.e., handle) is one thatis prepared and then added to the support material as opposed to beingformed or built up on the support material. Although all supportmaterials with linkers attached thereto described herein are not madeusing preformed handles, it is particularly desirable to do so. Thus,the methods of the present invention attach a preformed divalent linkerto a support material either before or after it is attached to anamine-containing organic group.

Advantageously, in preferred embodiments, the method of preparing thesupport material of Formula I is carried out under relatively mildconditions. Preferably, the step of attaching the amine-containingorganic group to the divalent linker, and more preferably both the stepof attaching the amine-containing organic group to the divalent linkerand the step of attaching the divalent linker to the support material,is carried out at a temperature of no greater than 35° C. This reducesthe chances of racemization of any chiral groups, such as chiral aminoacid residues. The temperature at which either or both of these steps iscarried out is more preferably about 0-30° C., and most preferably about20-25° C. Typically, either or both of these steps is carried out for nogreater than about 30 hours each, preferably for no greater than about10 hours, more preferably for no greater than about 5 hours, and mostpreferably for no greater than about 2 hours each.

In the amine-containing organic group (i.e., —N(Y)—C(R¹)(R²)(R³)), thegroups R¹, R², and R³ are each independently H or an organic group. Theycan be a wide variety of organic groups, such as alkyls, aryls,heterocyclics, etc. Typically, at least one of R¹, R², and R³ is anamino acid side chain, which can be proteinogenic or non-proteinogenicamino acid side-chains.

Preferably, R¹, R², and R³ are each independently H or a (C₁-C₁₈)alkylgroup, a (C₆-C₈)aryl group, a (C₁-C₁₈)alk(C₆-C₁₈)aryl group, a(C₅-C₁₈)heterocyclic group, or a (C₁-C₁₈)alk(C₃-C₁₈)heterocyclic group.More preferably, at least one of R¹, R², and R³ is selected from thegroup consisting of a —CH₃, —CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH₂—CH(CH₃)₂,—(CH₂)_(n)X (n=1-4), and —CH(CH₃)X group wherein X is selected from thegroup consisting of —OH, —OCH₃, —NO₂, —NH₂, —SH, —SCH₃, —C(O)OH,—C(O)NH₂, —C₆H₅, —C6H₄OH, indoyl, imidazoyl, and protected derivativesthereof.

For certain embodiments of the support material of Formula I, R¹ and R²are each independently H or an organic group as defined above, and R³ isan organic group having a protecting group Z that is removable undermild conditions (e.g., moderate or weak acid, moderate or weak base,photolysis, thiolysis, palladium-catalyzed or rhodium-catalyzednucleophilic transfer, hydrogenation, or fluoridolysis). As used herein,moderate or weak acids are those with an H_(O) of −5 or higher, asdefined by J. P. Tam et al., in The Peptides, Vol. 9 (S. Udenfriend andJ. Meienhofer, Eds.), pp. 185-248, Academic Press, New York (1987).Examples of such acids include, but are not limited to, hydrochloric,acetic, dilute sulfuric, and trifluoroacetic acid. Moderate or weakbases are those having conjugate acids with a pka of of 15.0 or less.Examples of such bases include, but are not limited to, piperdine,morpholine, and 1,8-diazabicyclo[5.4.0]undec-7-ene.

Suitable protecting groups Z depend on the functionality of R³, whichcould include amines, carbonyls, hydroxyls, carboxylic acids, aldehydes,thiocarbonyls, etc. In preferred embodiments of the support material ofFormula I, R³ is selected from the group consisting of —C(O)OZ, —C(O)SZ,—C(S)OZ, —C(OZ)₂R⁴ (wherein R⁴ is an alkyl group, an aryl group, or anaralkyl group), and —C(OZ)₂H. Examples of suitable protecting groups Zinclude allyl, (C₁-C₄)alkyls, trityl, etc. Preferably, the protectinggroup Z is selected from the group consisting of methyl, t-butyl, andallyl moieties.

The protecting group Y of the support material of Formula I can be awide variety of protecting groups that can be removed using conditionsthat do not cleave the N—L bond, do not remove the linker from thesupport material, and do not adversely affect the compound (e.g., thepeptide) being formed on the support material. Thus, the linker L andthe protecting group Y are preferably chosen such that they can beremoved in an orthogonal fashion. An orthoganol protection scheme isdefined as one which makes use of two or more independent classes ofgroups, each one removed through a different chemical mechanism,allowing them to be removed in any order and in the presence of all theother classes.

Suitable protecting groups (Y) include, for example, those that can beremoved using a wide variety of known conditions. Preferably, theprotecting group Y is chosen such that it can be removed using mildconditions such as moderate or weak acid, moderate or weak base,photolysis, thiolysis, palladium- or rhodium-catalyzed nucleophilictransfer, hydrogenation, and fluoridolysis.

Examples of suitable protecting groups (Y) include, but are not limitedto: formyl; alkyl groups; aryl groups such as p-phenylbenzyl and9-phenylfluorenyl; alkenyl groups; aralkyl groups; aralkenyl groups;alkylcarbonyl groups such as acetyl (Ac) and derivatives of acetyl suchas acetoacetyl, mono-, di-, and tri-halogen substituted acetyl (e.g.,chloroacetyl, trichloroacetyl, trifluoroacetyl, etc.),o-nitrophenoxyacetyl, and phenylacetyl; arylcarbonyl groups such asbenzoyl and p-nitrobenzoyl; alkyloxycarbonyl groups such asmethoxycarbonyl, ethoxycarbonyl, isobutoxycarbonyl,1-adamantyloxycarbonyl, 1,1-dimethyl-2-cyanoethoxycarbonyl,1,1-dimethyl-2,2-dibromoethoxycarbonyl,1,1-dimethyl-2,2,2-trichloroethoxycarbonyl, diisopropylmethoxycarbonyl,2-iodoethoxycarbonyl, and 2-(trimethylsilyl)ethoxycarbonyl;aryloxycarbonyl groups such as m-nitrophenyloxycarbonyl andphenyloxycarbonyl; alkenylmethoxycarbonyl groups such asallyloxycarbonyl and 4-nitrocinnamyloxycarbonyl; alkenyloxycarbonylgroups such as vinyloxycarbonyl; aralkyloxycarbonyl groups such as1-methyl-1-phenylethoxycarbonyl, 1-methyl-1-(4-pyridyl)ethoxycarbonyl,di(2-pyridyl)methoxycarbonyl, 1-methyl-1-(4-biphenyl)ethoxycarbonyl, and9-fluorenylmethoxycarbonyl; cycloalkyloxycarbonyl groups such ascyclobutyloxycarbonyl, cyclohexyloxycarbonyl, cyclopentyloxycarbonyl,and 1-methyl-1-cyclohexyloxycarbonyl; alkylaminooxycarbonyl groups suchas N-hydroxypiperidinyloxycarbonyl; sulfenyl groups such aso-nitrophenylsulfenyl and 3-nitropyridinesulfenyl; and sulfonyl groupssuch as p-toluenesulfonyl and β-(trimethylsilyl)ethanesulfonyl.

The protecting group Y is preferably an N^(α)-amine protecting group.Examples of suitable N^(α)-amine protecting groups include, for example,Fmoc, Aloc, Boc, Ddz, Npys, Nvoc, Bpoc, Teoc, Trt, SES, allyl, and t-Bu.These abbreviations are defined in the Examples section below. Apreferred group of N^(α)-amine protecting groups include, Fmoc, Aloc,and Boc.

The linker L in the support material of Formula I can be a wide varietyof handles used in solid phase peptide synthesis. The linker L is abifunctional spacer that, on one end, incorporates features of asmoothly cleavable protecting group, and on the other end, a functionalgroup, often a carboxyl group, that can be activated to allow couplingto the functionalized support material. The linker can be a preformedlinker or handle or it can be prepared on the support material. Suitablelinker (L) examples include the PAL handle[5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid] and the XALhandle [5-(9-aminoxanthen-2-oxy)valeric acid]. Other suitable linkersinclude handles such as 4-(α-aminobenzyl)phenoxyacetic acid,4-(α-amino-4′-methoxybenzyl)phenoxybutyric acid,4-(α-amino-4′-methoxybenzyl)-2-methylphenoxyacetic acid,2-hydroxyethylsulfonylacetic acid, 2-(4-carboxyphenylsulfonyl)ethanol,and those disclosed in G. B. Fields et al., Synthetic Peptides: A User'sGuide, 1990, 77-183, G. A. Grant, Ed., W. H. Freeman and Co., New York.

A preferred group of linkers is of the following formula (Formula II):

wherein:

each U is independently selected from the group consisting of an alkylgroup, an alkoxy group, an aryl group, an alkoxyaryl group, an aralkylgroup, an aralkoxy group, an alkylthio group, an arylthio group, analkylamido group, an alkylsulfinyl group, an alkylsulfonyl group, analkylsulfoxide group, a halogeno group, and a nitro group, wherein anytwo U groups can be joined to form a ring; W is a functionalized spacergroup for anchoring the linker to the support material; R⁵ and R⁶ areeach independently H, an alkyl group, or an aryl group; and x=0-4.

Preferably, W is selected from the group consisting of—(CH₂)_(n)C(O)NH—, —O(CH₂)_(n)C(O)NH—, —NH(CH₂)_(n)C(O)NH—,—OC(O)(CH₂)_(n)C(O)NH—, —C(O)(CH₂)_(n)C(O)NH—, —C(O)O(CH₂)_(n)C(O)NH—,—NHC(O)(CH₂)_(n)C(O)NH—, —O(CH₂)C₆H₄C(O)NH—, —C(O)O(CH₂)C₆H₄C(O)NH—,—OC(O)C₆H₄C(O)NH—, —OC(O)(CH₂CH₂O)_(n)C(O)NH—, —O(CH₂CH₂O)_(n)C(O)NH—,—NH(CH₂CH₂O)_(n)C(O)NH—, and —NHC(O)(CH₂CH₂O)_(n)C(O)NH— wherein n=1-18.

The present invention also provides a useful method of synthesizing anorganic compound using an aldehyde-functionalized support material. Thisaldehyde-functionalized support material has the following formula(Formula III):

wherein:

Ŝ represents a support material; V is NH, S or O; T is O, NH, NHC(O)R⁴,or S, wherein R⁴ is is an alkylene group, an arylene group, or anaralkylene group; R⁷ is an alkylene group, an arylene group, or anoxyalkylene group; each U is independently selected from the groupconsisting of an alkyl group, an alkoxy group, an aryl group, analkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthiogroup, an arylthio group, an alkylamido group, an alkylsulfinyl group,an alkylsulfonyl group, an alkylsulfoxide group, a halogeno group, and anitro group, wherein any two U groups can be joined to form a ring;x=0-4; and n=1-18. Preferably, R¹ is an alkylene group and n=1-4.

An amine-containing organic group is then typically attached to thealdehyde functionality under reducing conditions. Typically, this iscarried out at a temperature of no greater than 35° C. for no greaterthan about 10 hours. As used herein, reducing conditions includeNaBH₃CN, NABH₄, cat.H₂, and NaBH(OAc)₃. A second organic group, whichmay be a protected amino acid, is then attached to the N atom of theamine-containing group to build an organic compound. This is typicallycarried out in a nonprotic solvent selected from the group consisting ofCH₂Cl₂, ClCH₂CH₂Cl, tetrahydrofuran, CH₃CN, toluene, pyridine, dioxane,diethyl ether, and benzene. The organic compounds prepared may or maynot be a peptide, which may or may not be cyclic, or have a broad rangeof functional groups on the peptide being formed. For example, themethod of the present invention can provide a terminal carboxylic acidgroup, ester group, aldehyde group, thioacid group, and thioester group.

The present invention also provides a kit that includes thealdehyde-functionalized support material of Formula III and instructionsfor preparing an organic compound on the aldehyde-functionalized supportmaterial.

The present invention also provides a preformed linker having anamine-containing organic group attached thereto, of the formula (FormulaIV):

wherein:

L represents a divalent linker; Q represents a group selected from thegroup consisting of C(O)OH, C(O)OPfp, C(O)F, C(O)Br, C(O)Cl, OH, Br, Cl;Y represents H or a protecting group; R¹ and R² are each independently Hor an organic group; and R³ is an organic group having a protectinggroup Z that is removable under mild conditions.

The linker is attached to functionalized support materials or to spacerarms attached to the support materials. Typically, the support materialincludes hydroxyl, carboxyl, or amino functional groups, although otherfunctional groups such as thiol, halogens, and silyl are possible. Thesupport material can also include a spacer. Typically, a spacer is analkyl chain (preferably a (C₁-C₂₀)alkyl chain) substituted with an aminogroup and a carboxyl group.

A variety of functionalized support materials can be used. They can beof inorganic or organic materials and can be in a variety of forms(e.g., membranes, particles, spherical beads, fibers, gels, glasses,etc.). Examples include, porous glass, silica, polystyrene,polydimethylacrylamides, cotton, paper, and the like. Examples ofsuitable support materials are described by G. B. Fields et al., Int. J.Peptide Protein Res., 1990, 35, 161-214 and G. B. Fields et al.,Synthetic Peptides: A User's Guide, 1990, 77-183, G. A. Grant, Ed., W.H. Freeman and Co., New York. Functionalized polystyrene, such asamino-functionalized polystyrene, aminomethyl polystyrene, aminoacylpolystyrene, p-methylbenzhydrylamine polystyrene, or polyethyleneglycol-polystyrene resins can be used for this purpose. Polyethyleneglycol-polystyrene (PEG-PS) graft copolymers functionalized with aminogroups are particularly useful support materials. Suitable PEG-PS resinsare available from PerSeptive BioSystems (Framingham, Mass.) and aredescribed in U.S. Pat. No. 5,235,028 (Barany et al.).

The support materials of the present invention can be prepared byattaching the linker to the support material and then attaching theamine-containing organic group, e.g., amino acid or peptide. Theattachment reaction of the linker to the support material can be carriedout using standard coupling methods, e.g., acylations or alkylations, asdisclosed in, for example, F. Albericio et al., J. Org. Chem., 1990, 55,3730-3743, or alkylations. For example, acylations promoted byN,N′-dicyclohexylcarbodiimide (DCC), or N,N′-diisopropylcarbodiimide(DIPCDI) plus 1-hydroxybenzotriazole (HOBt), orbenzotriazoylyl-N-oxytris(dimethylamino)phosphonium hexafluorophosphate(BOP) plus 1-hydroxybenzotriazole (HOBt). Typically, one equivalent ofthe linker is used for each equivalent of functional group, e.g., aminogroup, present on the support. After attaching the amine-containinggroup to the linker, the protecting group can be added.

Alternatively, the linker, amino acid, and protecting group (if used)can be combined to form an optionally protected amino acid preformedlinker, which is then attached to the support material. For example, inone embodiment of the present invention, an aldehyde precursor of thePAL handle [5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid],which is disclosed in Albericio and Barany, Int. J. Pept. Protein Res.,1987, 30, 206-216, can be coupled through a reductive aminationprocedure to the α-amine of the prospective C-terminal amino acid orother amine-containing compound, which can be protected as a tert-butyl,methyl, trityl, or allyl ester, or modified to a dimethyl acetal. Theresultant intermediates, all secondary amines, can be treated withFmoc-Cl or Fmoc-OSu to give the corresponding protected amino acid (orother amine-containing compound) preformed handles in 40-70% yields.

The invention will be further described by reference to the followingdetailed examples. These examples are offered to further illustrate thevarious specific and illustrative embodiments and techniques. It shouldbe understood, however, that many variations and modifications may bemade while remaining within the scope of the present invention.

EXAMPLES

General Procedures. Protected Fmoc- and Boc-amino acid derivatives,Fmoc-OSu, Fmoc-Cl, HATU, PyAOP, BOP, and HOAt were from the BiosearchDivision of PerSeptive Biosystems (Framingham, Mass.), Bachem Bioscience(Philadelphia, Pa.) or Advanced ChemTech (Louisville, Ky.). MBHA resinsfor peptide synthesis were from NovaBiochem (San Diego, Calif.). PEG-PSresins (with Nle “internal reference” amino acid) were from theBiosearch Division of PerSeptive BioSystems. Amino acid esters (glycinemethyl ester, hydrochloride salt; phenylalanine methyl ester,hydrochloride salt; alanine tert-butyl ester, hydrochloride salt;leucine tert-butyl ester, hydrochloride salt) were from AdvancedChemTech or Bachem Bioscience. Piperidine, TFA, DIEA, HOBt, DMF, NaHCO₃,MgSO₄ (anhydrous), Na₂SO₄ (anhydrous), diethyl ether (HPLC grade), and 3Å molecular sieves were from Fisher (Pittsburgh, Pa.). DIPCDI,diethyldithiocarbamic acid (sodium salt), allyl bromide,2,2-dimethoxyethylamine, 2,4-dihydroxybenzaldehyde,4-formyl-2,6-dimethylphenol, ethyl 5-bromovalerate, and sodiumcyanoborohydride were from Aldrich (Milwaukee, Wis.). Methanol(anhydrous), dioxane, ethyl acetate (anhydrous), acetic anhydride, andhexane (HPLC grade) were from EM Science (Gibbstown, N.J.). CsHCO₃ wasfrom Alfa (Ward Hill, Mass.). 5-(4-Formyl-3,5-dimethoxyphenoxy)valericacid (PALdehyde) and 5-(2-formyl-3,5-dimethoxyphenoxy)valeric acid wereprepared according to Albericio et al., J. Org. Chem., 1990, 55,3730-3743. A mixture of 5-(4-formyl-3,5-dimethoxyphenoxy)butyric acidand 5-(2-formyl-3,5-dimethoxyphenoxy)butyric acid (o,p-PALdehyde),prepared essentially as described by Albericio and Barany, Int. J. Pept.Protein Res., 1987, 30, 206-216, was provided by the Biosearch Divisionof PerSeptive BioSystems. Organic solvent extracts were dried overanhydrous MgSO₄ or Na₂SO₄, followed by solvent removal at reducedpressures and <40° C. Silica gel chromatography was performed withSilica Gel 60 (230-400 mesh) from EM Science, unless otherwise stated.Elemental analyses were conducted by M-H-W Laboratories (Phoenix,Ariz.). Melting points were determined on a Büichi apparatus and areuncorrected.

Thin-layer chromatography was performed on either Polygram SIL G/UV₂₅₄plates (250 mm, 40×80 mm, Macherey-Nagel) or Kieselgel 60 F₂₅₄ (0.2 mm,40×80 mm, EM Science). Spots were visualized by UV. Analytical HPLC wasperformed using a Waters (Milford, Mass.) Nova Pak analytical C-18reversed phase-column (0.39×15 cm) Nucleosil (Macherey-Nagel) C-18column (0.46×25 cm) on a Waters system configured with a 600E SystemController, a 625 Pump, a 700 Satellite WISP autoinjector, and a 996Photodiode Array Detector or a Vydac (Hesperia, Calif.) analytical C-18reversed phase-column (0.46×25 cm) on a Beckman System configured withtwo 112 pumps and a 165 Variable Wavelength Detector. Low resolutionfast atom bombardment mass spectroscopy (FABMS) was carried out inglycerol-H₂O or 3-nitrobenzyl alcohol (MNBA) matrices on a VG Analytical7070E-HF low-resolution double-focusing mass spectrometer equipped witha VG 11/250 data system, operated at a resolution of 2000. Electrospraymass spectrometry was performed on a Perkin Elmer Sciex API III triplequadrupole mass spectrometer equipped with an ionspray interface.Parameters were: ionspray voltage 5000V, interface temperature 55° C.,potential on first quadrupole 30V, orifice voltage varied from 50 to150V. The curtain gas flow (N₂) and the nebulizer gas (ultrapure air)were set at 0.8-1.0 L/minutes. Molecular masses were calculated with theSciex MacSpec 3.22 programm. MALDI-TOF mass spectrometry was performedon a Kompact Maldi I (Kratos Analytical). ¹H NMR spectroscopy wasperformed on Varian VXR 300 and VXR 500 instruments operating at 300 and500 MHz, respectively. Chemical shifts (δ) are expressed in parts permillion downfield from TMS. Coupling constants, in parentheses, areexpressed in hertz. Amino acid analyses were carried out on a Beckman6300 analyzer. Samples were hydrolyzed with 6 N HCl in propionic acid at160° C., for 1 hour (condition A), or with an added 1 drop of phenol at155° C., for 3 hours (condition B). Reported cleavage yields are basedon amino acid ratios with respect to Ile “internal reference” amino acidof recovered peptidyl-resins after TFA treatment.

Example 1N-^(α)-[4-(carboxylbutyloxy)-2,6-dimethoxybenzyl]-N^(α)-(9-fluorenylmethoxycarbonyl)glycinemethyl ester

5-(4-Formyl-3,5-dimethoxyphenoxy)valeric acid (140 mg, mw 282.3, 0.5mmol), glycine methyl ester, hydrochloride salt, (63 mg, mw 125.6, 0.5mmol) and NaBH₃CN (31 mg, mw 62.84, 0.5 mmol) were combined in a 50 mLround-bottom flask and suspended in methanol (8 mL) and stirred at 25°C. for 60 minutes. The suspension was concentrated to dryness in vacuoand the remaining oil was resuspended in dioxane-water (1:1, 4 mL).Solid NaHCO₃ (126 mg, mw 84.0, 1.5 mmol) was added and the suspensionwas cooled in an ice-bath. Fmoc-OSu (253 mg, mw 337.3, 0.75 mmol)dissolved in dioxane (2 mL) was added to the suspension. Stirring wascontinued for 1 hour while cooling in an ice-bath, and at 25° C.overnight. The pH was then adjusted to 9 by addition of saturatedaqueous NaHCO₃. The suspension was diluted with water (20 mL) and washedwith diethyl ether (2×25 mL). The phase separations were slow and theether layer remained cloudy. The aqueous layer was acidified to pH 3with 4 N aqueous HCl and extracted with ethyl acetate (3×30 mL). Thecombined organic phases were dried over MgSO₄, filtered and concentratedto dryness in vacuo to provide an oil (278 mg, 96%).

¹H NMR (500 MHz, DMSO-d₆, 368 K) δ11.61 (broad s), 7.85 (d, J=6.9; 2H),7.7-7.5, m; 2H), 7.40 (t, J=7.4; 2H), 7.32 (m; 2H), 6.22 (s; 2H), 4.48(broad s; 1H), 4.36 (d, J=6.7; 2H), 4.32 (dd, J=10.8, 6.9; 1H), 4.26(broad s; 1H), 4.01 (t, J=6.2; 2H), 3.78-3.72 (m; 2H), 3.70 (s; 6H),3.68 (s; 1H), 3.64 (s; 1H), 3.59 (s; 3H), 3.58 (s; 1H), 2.60 (s; 1H),2.29 (t, J=7.1; 2H), 1.75 (q, J=13.7, 6.5; 2H), 1.70 (q, J=14.2, 7.4;2H).

FABMS m/z calcd for C₃₂H₃₅NO₉: 577.6, found: 577.5 [M⁺.].

Example 2N^(α)-[4-(carboxylbutyloxy)-2,6-dimetboxybenzyl]-N^(α)-(9-fluorenylmethoxycarbonyl)glycineallyl ester

Preparation of H-Gly-OAl trifluoroacetate salt: N^(α)-Boc-Gly-OH (8.76g, mw 175.2, 50 mmol) was suspended in water-dioxane (1:4, 100 mL).CsHCO₃ (10.7 g, mw 193.92, 55 mmnol), dissolved in water (20 mL) wasadded over 5 minutes; 10 minutes later the suspension was concentratedto dryness in vacuo. The remaining foam was suspended in DMF (80 mL),stirred with allyl bromide (4.8 mL, mw 121.0, ρ 1.40, 55 mmol) at 25° C.for 14 hours, and then concentrated to dryness in vacuo. The solid wassuspended in ethyl acetate (300 mL) and extracted with 10% aqueousNaHCO₃ (3×150 mL). The aqueous phase was backwashed with ethyl acetate(100 mL) and the combined organic phases were dried over MgSO₄,filtered, and concentrated in vacuo to an oil; Yield 10.6 g. The crudeproduct was purified by vacuum liquid chromatography (over TLC gradesilica gel 60 G using ethyl acetate-hexane [1:4] as eluent); Yield 9.3g. The oil was treated with TFA-CH₂Cl₂ (1:1, 50 mL) for 1 hour and thenconcentrated in vacuo to an oil, which was washed with diethyl ether(3×50 mL) to give off-white crystals (7.74 g, 68%).

Preparation ofN^(α)-[4-(carboxylbutyloxy)-2,6-dimethoxybenyl]-N^(α)-(9-fluorenylmethoxycarbonyl)glycineallyl ester: 5-(4-Formyl-3,5-dimethoxyphenoxy)valeric acid (140 mg, mw282.3, 0.50 mmol) and glycine allyl ester, trifluoroacetate salt, (138mg, mw 229.2, 0.6 mmol) were dissolved in methanol (5 mL) in a 25 mLround-bottom flask, and stirred at 25° C. for 10 minutes. NaBH₃CN (47mg, mw 62.84, 0.75 mmol) was added and the suspension stirred at 25° C.for 60 minutes. The suspension was concentrated to dryness in vacuo andthe remaining oil was resuspended in dioxane-saturated aqueous NaHCO₃(2:1, 15 mL) and the suspension was cooled in an ice-bath. Fmoc-OSu (223mg, mw 337.3, 0.66 mmol) was dissolved in dioxane (2 mL) and added tothe suspension. Stirring was continued for 1 hour while cooling in anice-bath at 5° C. for 14 hours, and then at 25° C. for 2 hours. Thesuspension was diluted with water (20 mL) and washed with diethyl ether(2×25 mL). The aqueous layer was acidified to pH 3 by addition of 0.5 Naqueous HCl and extracted with ethyl acetate (3×20 mL). The combinedorganic phases were dried over MgSO₄, filtered and concentrated todryness in vacation to provide an oil (286 mg, 95%).

¹H NMR (500 MHz DMSO-d₆, 294 K) δ12.05 (broad s), 10.48 (broad s), 7.87(d, J=7.3; 3H), 7.76 (t, J=6.1; 1H), 7.68 (t, J=7.3; 2H), 7.40 (t,J=7.3; 3H), 7.31 (t, J=7.5; 3H), 6.19 (broad s, 1H), 5.87 (m; 1H), 5.29(d, J=17.1; 1H), 5.19 (d, J=10.7; 1H), 4.57 (d, J=4.5; 2H), 4.28 (m;2H), 4.22 (t, J=6.8; 1H), 3.8-3.1 (m; ˜20H), 2.17 (broad s; 2H), 1.49(broad s, 4H).

FABMS m/z calcd for C₃₄H₃₇NO₉: 603.25, found: 603.2 [M⁺.].

Example 3N^(α)-[4-(carboxylbutyloxy)-2,6-dimethoxybenzyl]-N^(α)-(9-fluorenylmethoxycarbonyl)phenylalaninemethyl ester

5-(4-Formyl-3,5-dimethoxyphenoxy)valeric acid (560 mg, mw 282.3, 2.0mmol), phenylalanine methyl ester, hydrochloride salt, (431 mg, mw215.7, 2.0 mmol) and NaBH₃CN (189 mg, mw 62.8, 3.0 mmol) were suspendedin methanol (6 mL) in a 25 mL round-bottom flask and stirred at 25° C.for 60 minutes. The suspension was concentrated to dryness in vacuo andthe remaining oil was resuspended in dioxane-water (1:1, 12 mL). SolidNaHCO₃ (504 mg, mw 84.01, 6 mmol) was added and the suspension cooled inan ice-bath. Fmoc-Cl (569 mg, mw 258.7, 2.2 mmol) was dissolved indioxane (4 mL) and added to the suspension. Stirring was continued for 1hour while cooling on an ice-bath, and then at 25° C. for 26 hours. Thesuspension was diluted with water (50 mL) and washed with hexane (2×50mL). The phase separations were slow and the hexane layer remainedcloudy. The aqueous layer was acidified to pH 3 by addition 0.5 Naqueous HCl (3 mL) and extracted with ethyl acetate (3×50 mL). The firstseparation was left for 2 hours, the second overnight. The combinedorganic phases were dried over MgSO₄, filtered and concentrated todryness in vacuo to provide an oil (1.14 g, 84%).

¹H NMR (500 MHz, DMSO-d₆, 368 K) δ11.33 (broad s), 7.84 (d, J=7.0; 2H),7.66 (t, J=9.5, 7.5; 2H), 7.39 (q, J=14.0, 7.0; 2H), 7.32 (t, J=7.3;2H), 7.09 (broad s, 3H), 6.78 (broad s, 2H), 6.10 (s, 2H), 4.45 (dd,J=10.0, 5,8; 1H), 4.30 (m; 1H), 4.26 (t, J=6.0; 2H), 4.16 (d, J=14.6;1H), 3.98 (t, J=6.5; 2H), 3.87 (broad s, 1H), 3.61 (s, 6H), 3.41 (broads, 3H), 2.28 (t, J=7.3; 2H), 1.74 (m, 2H), 1.69 (m, 2H).

FABMS m/z calcd for C₃₉H₄₁NO₉: 667.28, found: 667.3 [M⁺.].

Example 4N^(α)-[4-(carboxylbutyloxy)-2,6-dimethoxybenzyl]-N^(α)-(9-fluorenylmethoxycarbonyl)alaninetert-butyl ester

5-(4-Formyl-3,5-dimethoxyphenoxy)valeric acid (701 mg, mw 282.3, 2.5mmol), alanine tert-butyl ester, hydrochloride salt, (454 mg, mw 181.7,2.5 mmol) and NaBH₃CN (236 mg, mw 62.8, 3.75 mmol) were suspended inmethanol (10 mL) in a 25 mL round-bottom flask and stirred at 25° C. for60 minutes. The suspension was concentrated to dryness in vacuo and theremaining oil was resuspended in dioxane-water (1:1, 4 mL). Solid NaHCO₃(630 mg, mw 84.0, 7.5 mmol) was added, and the suspension was cooled inan ice-bath. Fmoc-Cl (971 mg, mw 258.7, 3.75 mmol) was dissolved indioxane (3 mL) and added to the suspension. Stirring was continued for Ihour while cooling in an ice-bath and at 25° C. overnight. The pH wasthen adjusted to 9 by addition of saturated aqueous NaHCO₃ (6 mL). Thesuspension was diluted with water (20 mL) and washed with diethyl ether(2×35 mL). The aqueous layer was acidified to pH 2 with 1.5 N aqueousHCl and extracted with ethyl acetate (3×20 mL). The combined organicphases were dried over MgSO₄, filtered and concentrated to dryness invacuo to provide an oil (905 mg, 57%).

¹H NMR (500 MHz, DMSO-d₆, 368K) δ11.1 (broad s; 1H), 7.85 (d, J=7.6;2H), 7.66 (t, J=6.6; 2H), 7.40 (t, J=7.5; 2H), 7.31 (ddt, J=7.5, 2.8,1.1; 2H), 6.23 (s; 2H), 4.58 (d, J=13.7; 2H), 4.44 (m; 1H), 4.40 (d,J=13.7; 2H), 4.28-4.24 (m; 2H), 4.02 (t, J=6.5; 2H), 3.75 (d, J=7.5;1H), 3.72 (s; 6H), 3.66 (d, J=7.0; 1H), 2.29 t, J=9 7.2; 2H), 1.97 (s;2H), 1.75 (m; 2H), 1.70 (m; 2H), 1.32 (s; 7H), 1.09 (d, J=6.7; 2H).

FABMS m/z calcd for C₃₆H₄₃NO₉: 633.3, found: 633.3 [M⁺.].

Example 5N^(α)-[4-(carboxylbutyloxy)-2,6-dimethoxybenzyl]-N^(α)-(9-fluorenylmethoxycarbonyl)-2,2-dimethoxyethylamine

5-(4-Formyl-3,5-dimethoxyphenoxy)valeric acid (560 mg, mw 282.3, 2.0mmol), 2,2-dimethoxyethylamine (261 μL, mw 105.1, ρ 0.97, 2.4 mmol) andmolecular sieves 3 Å (100 mg) were combined in a 25 mL round-bottomflask. Methanol (10 mL) was added, and the reaction mixture was refluxedwith magnetic stirring for 90 minutes. After cooling to 25° C., NaBH₃CN(189 mg, mw 62.8, 3.0 mmol) was added in two portions over 5 minutes andstirring continued for 60 minutes. The suspension was concentrated todryness in vacuo and stored overnight at −20° C. The remaining oil wasresuspended in dioxane-water (3:2, 25 mL), and solid NaHCO₃ (840 mg, mw84.0, 10 mmol) was added. The suspension was cooled in an ice-bath.Fmoc-OSu (890 mg, mw 337.3, 2.64 mmol) was dissolved in dioxane (8 mL)with gentle heating and added from an addition funnel over 15 minutes tothe suspension. Stirring was continued for 2 hours while cooling on anice-bath and then for 2 hours at 25° C. The suspension was diluted withwater (15 mL) and washed with diethyl ether (30 mL). The aqueous layerwas diluted with water (50 mL) and extracted with diethyl ether (50 mL).The aqueous layer was acidified to pH 3 by addition of 0.5 N aqueous HCland extracted with ethyl acetate (4×50 mL). The combined organic phaseswere dried over MgSO₄, filtered and concentrated to dryness in vacuo toan oil. The oil was stored at −20° C. overnight. The oil was thendissolved in diethyl ether (100 mL) at 25° C. and hexane was added untilcloudiness occurred. Storage at −20° C. overnight resulted in theformation of crystals together with some oil. The mixture wasconcentrated to dryness in vacuo, and suspended in diethyl ether. Afterconcentration to 50 mL in vacuo hexane was added until the onset ofcloudiness. It was stored at −20° C. overnight after which crystals hadformed (503 mg, 42%).

¹H NMR (500 MHz, DMSO-d₆, 294 K) δ12.05 (broad s; 1H), 7.85 (t, J=7.7;2H), 7.65 (broad s; 2H), 7.39 (q, J=14.3, 7.3; 2H), 7.31 (t, J=7.5; 2H),6.21 (broad s; 1H), 6.18 (broad s; 1H), 4.47 (m; 2H), 4.26 (t, J=˜6;2H), 3.97 (d, J=6.1; 2H), 3.67 (s; 6H), 3.21 (s; 3H), 3.04 (s; 3H), 2.28(t, J=7.3; 2H), 1.70 (m; 2H), 1.65 (m; 2H).

FABMS m/z calcd for C₃₃H₃₉NO₉: 593.67, found: 593.1 [M⁺.], 592.3 [M−H⁻].

Example 6N^(α)-[2-(carboxylbutyloxy)-4,6-dimethoxybenzyl]-N^(α)-(9-fluorenylmethoxycarbonyl)glycineallyl ester

5-(²-Formyl-3,5-dimethoxyphenoxy)valeric acid (2.80 g, mw 282.3, 10.0mmol), glycine allyl ester, trifluoroacetate salt, (2.15 g, mw 229.2,9.4 mmol; prepared as in Example 2) and NaBH₃CN (0.95 g, 62.8, 15 mmol)were suspended in methanol (45 mL) in a 50 mL round-bottom flask andstirred at 25° C. for 60 minutes. The suspension was concentrated todryness in vacuo in a 250 mL round-bottom flask and the remaining oilwas resuspended in dioxane-water (1:1, 16 mL). Solid NaHCO₃ (2.6 g, mw84.0, 30 mmol) was added and the suspension was cooled in an ice-bath.Fmoc-Cl (2.9 g, mw 258.7, 11.0 mmol) dissolved in dioxane (approximately4 mL) was added to the suspension and stirring continued for 1 hourwhile cooling in an ice-bath, and at 25° C. overnight. The suspensionwas diluted with water (20 mL), the pH was adjusted to 9 with saturatedaqueous NaHCO₃, and then extraction was carried out with diethyl ether(2×25 mL). The aqueous layer was acidified to pH 3 by addition of 1.5 Naqueous HCl (10 mL) and extracted with ethyl acetate (3×50 mL). Thecombined organic phases were dried over MgSO₄, filtered and concentratedto dryness in vacuo to provide an oil (4.31 g, 76%).

¹H NMR (500 MHz, DMSO-d₆, 368 K) δ7.85 (d, J=7.6; 2H), 7.80 (d, J=7.6;2H), 7.40 (dt, J=7.5, 1.1; 2H), 7.33 (dt, J=7.5, 1.1; 2H), 6.21 (s; 1H),6.19 (s; 1H), 5.89 (m; 1H), 5.28 (dq, J=17.2, 3.1, 1.5; 1H), 5.19 (dq,J=10.5, 2.8, 1.4; 1H), 4.52 (dt, J=5.5, 1.4; 2H), 3.96 (t, J=6.2; 2H),3.75 (s; 6H), 3.67 (s; 2H), 2.28 (t, J=7.1; 2H), 1.75 (dd, J=13.3, 6.6;2H), 1.70 (dd, J=14.8, 7.9; 2H).

FABMS m/z calcd for C₃₄H₃₇NO₉: 603.25, found: 603.2 [M⁺.], 648.4[M−H+2Na].

Example 7N^(α)-[4-(carboxylbutyloxy)benzyl]-N^(α)-(9-fluorenylmethoxycarbonyl)leucinetert-butyl ester

4-(4-Formyl-phenoxy)butyric acid (104 mg, mw 208.2, 0.50 mmol), leucinetert-butyl ester, hydrochloride salt, (112 mg, mw 223.8, 0.50 mmol) andNaBH₃CN (47 mg, mw 62.8, 0.75 mmol) were suspended in methanol (5 mL) ina 25 mL round-bottom flask stirred at 25° C. for 7 hours. The suspensionwas concentrated to dryness in vacuo and the remaining oil wasresuspended in dioxane-water (1:1, 4 mL), and solid NaHCO₃ (126 mg, mw84.0, 1.5 mmol) added. The suspension was cooled in an ice-bath. Fmoc-Cl(142 mg, mw 258.7, 0.55 mmol) was dissolved in dioxane (2 mL) and addedto the suspension. Stirring was continued for 1 hour while cooling in anice-bath, and at 25° C. for 20 hours. The suspension was diluted withwater (20 mL), which caused an emulsion to form. The emulsion wasconcentrated in vacuo at 40° C. to remove dioxane, and then extractedwith hexane (2×25 mL). The phase separations were slow. The aqueouslayer was acidified to pH 3 by addition of 0.5 N aqueous HCl andextracted with ethyl acetate (3×25 mL). The combined organic phases weredried over MgSO₄, filtered and concentrated to dryness in vacuo toprovide an oil (64 mg, 21%).

¹H NMR (500 MHz, DMSO-d₆, 294 K) δ12.12 (broad s; 1H), 7.84 (d, J=7.3;2H), 7.63 (t, J=7.3; 1H), 7.53 (t, J=10.1, 7.9; 1H), 7.38 (m; 2H),7.32-7.22 (m; 2H), 7.08 (d, J=7.9), 6.85 (d, J=8.2; 1H), 6.79 (d, J=8.2;1H), 6.70 (d, J=8.2; 1H), 4.53 (d, J=5.2; 2H), 4.24 (t, J=4.9; 1H),4.16-4.10 (m; 1H), 3.95 (m; 1H), 3.92 (t, J=6.5; 2H), 2.35 (m; 2H), 1.90(m; 2H), 1.51 (m; 1H), 1.26 (s, 9H), 0.68 (d, J=6.4; 1.5H), 0.62 (d,J=6.4; 1.5H), 0.49 (d, J=6.4; 1.5H), 0.44 (d, J=6.1; 1.5H).

FABMS m/z calcd for C₃₆H₄₃NO₇: 601.30, found: 602.3 [MH⁺].

Example 8 Coupling ofN^(α)-[4-(carboxylbutyloxy)-2,6-dimethoxybenzyl]-N^(α)-(9-fluorenylmethoxycarbonyl)-2,2-dimethoxyethylamine,Fmoc-BAL(glycinal dimethyl acetal)-OH, to PEG-PS

N^(α)-[4-(carboxylbutyloxy)-2,6-dimethoxybenzyl]-N^(α)-(9-fluorenylmethoxycarbonyl)-2,2-dimethoxyethylamine(214 mg, mw 593.7, 0.36 mmol), BOP (160 mg, mw 442.3, 0.36 mmol), HOBt(49 mg, mw 135.1, 0.36 mmol), were dissolved in DMF (3 mL). After 5minutes, this solution was added to neutralized PEG-PS resin (0.18mmol/g, 1.00 g) and reaction was carried out at 25° C. overnight. Theresin was washed with DMF (5×2 mL) and CH₂Cl₂ (5×2 mL). A solution ofacetic anhydride-CH₂Cl₂ (1:3, 4 mL) was added and a capping reaction wascarried out at 25° C. for 1 hour. The resin was washed with CH₂Cl₂ (10×2mL) and dried in vacuo at 25° C.

Example 9 Synthesis of model peptide aldehydeH-Ala-Leu-Ala-Lys-Leu-Gly-Gly-H, (SEQ ID NO: 1)

Manual chain assembly was carried out starting with Fmoc-BAL(glycinaldimethyl acetal)-PEG-PS resin (200 mg) prepared according to Example 8.Wash volumes were 2 mL. Side-chain protection for Lys was provided byBoc. Fmoc removal was accomplished with piperidine-DMF (1:4; 5minutes+25 minutes), followed by washes with DMF (5×2 minutes).N-Fmoc-Gly-OH (0.30 mmol, 89 mg) was dissolved in CH₂Cl₂-DMF (5:1, 1.2mL) and a solution of DCC (31 mg, mw 206.3, 0.15 mmol) in CH₂Cl₂ (0.5mL) was added. After 15 minutes a white precipitate was removed byfiltration and the solution added to the resin. CH₂Cl₂ was added to givea total volume of 2 mL. The remaining amino acids were coupled by inturn dissolving each N^(α)-Fmoc-amino acid (0.128 mmol) in DMF (1.5 mL)with BOP (43 mg, 442.3, 0.096 mmol), HOBt (13 mg, 135.1, 0.096 mmol),and DIEA (25 μl, mw 129.3, ρ 0.74, 0.145 mmol); after 5 minutes thesolution was added to the growing peptidyl-resin and reacted at 25° C.for 45 minutes. For cleavage, a portion (19 mg) of the peptidyl-resinwas treated first with piperidine-DMF (1:4) to remove Fmoc, followed bywashings and then TFA-H₂O (19:1) for 2 hours at 25° C. The filtrate fromthe cleavage reaction was collected and combined with TFA washes (3×2mL) of the cleaved peptidyl-resin, and the resultant solution wasconcentrated under a stream of N₂. The crude cleaved peptide wasprecipitated with methyl tert-butyl ether (2 mL), washed with diethylether (2×10 mL), and dried to give material that showed a single majorcomponent by analytical HPLC (4.6×250 mm column). ESMS m/z calcd: 612.4;found: 613.4 [MH⁺]. The amino acid composition of the hydrolyzed (A)peptidyl-resin was: Gly, 1.00; Ala, 1.98; Leu, 1.87; Lys, 0.91; Nle,4.14. The amino acid composition of the hydrolyzed (A) crude cleavedpeptide was: Gly, 1.00; Ala, 2.06; Leu, 1.86; Lys, 0.85.

Example 10 Preparation of H-BAL(Leu-OtBu)-Ile-PEG-PS by on-resinreductive amination

Coupling of N^(α)-Fmoc-Ile-OH to PEG-PS: PEG-PS (1.00 g, 0.21 mmol/g)was first treated with CH₂Cl₂ (5×0.5 minute), TFA-CH₂Cl₂ (4:6, 1minute+20 minutes), and then washed with CH₂Cl₂ (5×0.5 minute),neutralized by DIEA-CH₂Cl₂ (1:19, 3×1 minute), and washed with CH₂Cl₂(5×0.5 minute), DMF (5×0.5 minute). Fmoc-Ile-OH (3 equivalents), HOBt (3equivalents, 135.1) and DIPCDI (mw 126.2, ρ 0.81, 3 equivalents) weredissolved in DIMF (2 mL) and added to the resin. The reaction wascarried out at 25° C. for 2 hours, at which time the resin was negativeto the Kaiser ninhydrin test. The resin was washed with DMF (5×0.5minute) and CH₂Cl₂ (3×0.5 minute), and dried.

Coupling of 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid toH-Ile-PEG-PS: N^(α)-Fmoc-Ile-PEG-PS (0.1 g, 0.21 mmol/g) was firsttreated with piperidine-DMF (1:4, 3×1 minutes, 2×5 minutes, 2×1 minutes)and then washed with DMF (5×0.5 minute).5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid (PALdehyde; 24 mg, mw282.3, 4 equivalents), HATU (32 mg, mw 380.2, 4 equivalents) and DIEA(29 μL, mw 129.25, ρ 0.74, 8 equivalents) were dissolved in DMF. After 1minute, this solution was added to the resin and the coupling wasallowed to proceed at 25° C. for 2 hours, at which time the resin wasnegative to the Kaiser ninhydrin test. The resin was washed with DMF(5×0.5 minute), CH₂Cl₂ (3×0.5 minute), and dried in vacuo.

Preparation of H-BAL(Leu-O^(t)Bu)-Ile-Nle-PEG-PS by solid-phasereductive amination: Leucine tert-butyl ester, hydrochloride salt, (47mg, mw 223.8, 10 equivalents) and NaBH₃CN (13 mg, mw 62.8, 10equivalents) dissolved in DMF (0.4 mL) were added toPALdehyde-Ile-PEG-PS (100 mg) and the reaction was allowed to proceed at25° C. for 1 hour. The resin was then washed with DMF (5×0.5 minute),CH₂Cl₂ (3×0.5 minute), DMF (3×0.5 minute), piperidine-DMF (1:4, 3×1minutes), DMF (5×0.5 minute), and CH₂Cl₂ (3×0.5 minute).

Example 11 Synthesis of model peptide H-Tyr-Gly-Gly-Phe-Leu-OH,Leu-enkephalin (SEQ ID NO: 2)

Manual chain assembly was carried out starting withH-BAL(Leu-OtBu)-Ile-PEG-PS resin (100 mg) prepared according to Example10. Wash volumes were 2 mL. Side-chain protection for Tyr was providedby tBu. Fmoc removal was accomplished with piperidine-DMF (1:4; 3×1minutes, 2×5 minutes, 2×1 minutes), followed by washes with DMF (5×0.5minute). N^(α)-Fmoc-Phe-OH (10 equivalents, mw 387.4, 81 mg) wasdissolved in CH₂Cl₂-DMF (9:1, 0.5 mL) and DIPCDI (20 μL, mw 126.2, ρ0.81, 6 equivalents) was added. After 5 minutes, the solution was addedto the growing peptidyl-resin and the coupling was allowed to proceed at25° C. for 2 hours. The resin was subsequently washed with CH₂Cl₂ (5×0.5minute) and DMF (5×0.5 minute), and the coupling was repeated once. Theremaining amino acids were coupled by in turn dissolving eachN^(α)-Fmoc-amino acid (5 equivalents), DIPCDI (5 equivalents) and HOBt(5 equivalents) in DMF. Amino acid composition of the hydrolyzed (B)peptidyl-resin: Tyr, 0.85; Gly, 1.69; Phe, 0.85; Leu, 0.97; Ile, 1.00;Nle, 3.04.

The completed peptidyl-resin (50 mg) was treated first withpiperidine-DMF (1:4) to remove Fmoc, followed by washings with DMF (5×1minute) and CH₂Cl₂ (5×1 minute), and then cleaved with TFA-H₂O (19:1, 1mL) for 1 hour at 25° C. The filtrate from the cleavage reaction wascollected and combined with TFA washes (1 mL) of the cleavedpeptidyl-resin, and concentrated under a stream of N₂ to give materialthat showed a single major component by analytical HPLC (t_(R): 18.7minutes; 4.6×250 mm Vydac column, linear gradient over 30 minutes of0.1% TFA in CH₃CN and 0.1% aqueous TFA from 1:9 to 4:6, flow rate 1.0mL/minute, UV detection at 220 nm). The cleavage yield was 90% accordingto hydrolysis (B) of resin after cleavage: Tyr, 0.08, Gly, 0.17, Phe,0.08, Leu, 0.13, Ile, 1.00, Nle, 3.05. FABMS Wmz calcd: 555.2 ; found:556.2 [MH⁺].

Example 12 Synthesis of Leu-enkephalin C₈ ester (YGGFL-OOctyl)

The same procedures as described in Examples 10 and 11 were followed,except that PEG-PS was substituted with MBHA (0.57 mmol/g, 50 mg) andleucine tert-butyl ester, hydrochloride salt with leucine octyl ester,hydrochloride salt (80 mg, mw 279.8, 10 equivalents). N^(α)-Ddz-Phe-OH(110 mg, 10 equivalents), PyAOP (148 mg, mw 521.4, 10 equivalents), andDIEA (97 μL, mw 129.3, ρ 0.74, 20 equivalents) were dissolved in inCH₂Cl₂-DMF (9:1, 1 mL) and added to H-BAL(Leu-OOctyl)-Ile-MBHA and thereaction was carried out at 25° C. for 2 hours. The peptidyl-resin waswashed with CH₂Cl₂ (5×0.5 minute) and the coupling repeated twice (Atthis point, the amino acid composition of the hydrolyzed (B)peptidyl-resin was Leu, 0.98; Phe, 1.00). Treatment with TFA-H₂O-CH₂Cl₂(3:1:96, 6×1 minute) removed the Ddz group. Fmoc-Gly-OH (85 mg, mw297.3, 10 equivalents), PyAOP (148 mg, 10 equivalents), and DIEA (97 μL,20 equivalents) were dissolved in DMF (0.4 mL) and the solution added tothe resin. The reaction was allowed to proceed at 25° C. for 2 hours, atwhich time the Kaiser ninhydrin test was negative. The remaining aminoacids were coupled as in Example 11. Cleavage of the final peptide wasaccomplished with TFA-H₂O (19:1, 1 mL) at 25° C. for 90 minutes, and waspure by HPLC (98%, t_(R) 40.1 minutes; 4.6×250 mm Nucleosil column,linear gradient over 30 minutes of 0.1% TFA in CH₃CN and 0.1% aqueousTFA from 1:9 to 4:6, 10 minutes from 4:6 to 10:0, flow rate 1.0mL/minute, UV detection at 220 nm). The cleavage yield was 86% accordingto hydrolysis (B) of resin after cleavage. MALDI-TOF MS m/z calcd: 667.6; found: 668.9 [MH⁺], 692.6 [MNa⁺]. Amino acid composition of thehydrolyzed (B) final peptidyl-resin: Tyr, 0.97; Gly, 1.96; Phe, 0.98;Leu, 1.06; Ile, 1.00.

Example 13 Preparation of H-BAL(Ala-OAl)-Ile-MBHA by on-resin reductiveamination

Preparation of o,p-PALdehyde-Ile-MBHA: N^(α)-Fmoc-Ile-MBHA (0.57 mmol/g,50 mg) was first treated with piperidine-DMF (1:4, 3×1 minute+2×5minutes+2×1 minute) and then washed with DMF (5×0.5 minute). A mixtureof 5-(2-formyl-3,5-dimethoxyphenoxy)butyric acid and5-(4-formyl-3,5-dimethoxyphenoxy)butyric acid (o,p-PALdehyde; 31 mg, mw268.3, 4 equivalents), HATU (43 mg, mw 380.2, 4 equivalents), and DIEA(39 μL, mw 129.25, ρ 0.74, 8 equivalents) were dissolved in DMF (0.4mL). After 1 minute the solution was added to the resin and the reactionallowed to proceed at 25° C. for 2 hours, at which time the resin wasnegative to the Kaiser ninhydrin test. The resin was washed with DMF(5×0.5 minute), CH₂Cl₂ (3×0.5 minute), and dried in vacuo.

Preparation of H-BAL(Ala-OAl)-Ile-MBHA by solid-phase reductiveamination: H-Ala-OAl, hydrochloride salt (47 mg, mw 243.2, 10equivalents) and NaBH₃CN (18 mg, mw 62.8, 10 equivalents) were dissolvedin DMF (0.5 mL) and added to o,p-PALdehyde-Ile-MBHA (50 mg) and thereaction was allowed to proceed at 25° C. for 18 hours. The resin wasthen washed with DMF (5×0.5 minute), CH₂Cl₂ (3×0.5 minute), DMF (3×0.5minute), piperidine-DMF (1:4, 3×1 minute), DMF (5×0.5 minute), CH₂Cl₂(3×0.5 minute), and dried in vacuo.

Example 14 Synthesis of model linear peptideFmoc-Arg-DPhe-Pro-Glu-Asp-Asn-Tyr-Glu-Ala-Ala-OAl

Manual chain assembly was carried out starting withH-BAL(Ala-OAl)-Ile-MBHA resin (50 mg) prepared according to Example 13.Wash volumes were 2 mL. Side-chain protection was provided by Pmc (Arg),Trt (Asn), and tBu (Asp, Glu, Tyr). Fmoc removal was accomplished withpiperidine-DMF (1:4; 3×1 minute+2×5 minutes+2×1 minute), followed bywashes with DVIF (5×0.5 minute). N^(α)-Trt-Ala-OH (94 mg, mw 331.4, 10equivalents), PyAOP (148 mg, mw 521.4, 10 equivalents), and DIEA (97 μL,mw 129.3, ρ 0.74, 20 equivalents) were dissolved in DMF-CH₂Cl₂ (1:9),added to H-BAL(Ala-OAl)-Ile-MBHA (50 mg) and the reaction was carriedout at 25° C. for 2 hours. The peptidyl-resin was washed with CH₂Cl₂(5×0.5 minute), DMF (5×0.5 minute) and the coupling repeated. Treatmentwith TFA-H₂O-CH₂Cl₂ (1:1:98, 5×1 minute) removed the Trt group.Fmoc-Glu(OtBu)-OH (121 mg, mw 425.5, 10 equivalents), PyAOP (148 mg, 10equivalents) and DIEA (97 μL, 20 equiv) were dissolved in DMF (0.4 mL)and the solution added to the resin. The reaction was allowed to proceedat 25° C. for 2 hours, at which time the Kaiser ninhydrin test wasnegative. The remaining seven amino acid residues were consecutivelycoupled to the resin using Fmoc-AA-OH (5 equivalents), PyAOP (5equivalents) and DIEA (10 equivalents) in DMF. Amino acid composition ofthe hydrolyzed (B) peptidyl-resin was: Ala, 2.35; Arg, 0.90; Phe, 1.03;Pro, 0.87; Glu, 1.92; Asp, 1.93; Tyr, 1.01. The synthesis yield was 88%with respect to “internal reference” amino acid Ile. The final protectedpeptide was cleaved from the resin by treatment with TFA-Et₃SiH-H₂O(92:5:3) at 25° C. for 2 hours and was pure by HPLC (>65%, t_(R) 17.8minutes; 4.6×250 mm Nucleosil column, linear gradient over 30 minutes of0.1% TFA in CH₃CN and 0.1% aqueous TFA from 1:9 to 10:0, flow rate 1.0mL/minute, UV detection at 220 nm). MALDI-TOF MS: m/z calcd 1478.4;found 1474.8 [MH⁺].

Example 15 Cyclization of the peptideFmoc-Arg-DPhe-Pro-Glu-Asp-Asn-Tyr-Glu-Ala-Ala-OAl

The Fmoc and Al protected peptidyl-resin (10 mg) was washed with DMF(5×0.5 minute) followed by Pd(PPh₃)₄ (mw 1155.58, 5 equivalents) inDMSO-THF-0.5 N HCl-morpholine (2:2:1:0.1, 1.53 mL) at 25° C. for 3 hoursunder argon to cleave the C-terminal Al ester. The peptidyl-resin waswashed with THF (3×2 minutes), DMF (3×2 minutes), CH₂Cl₂ (3×2 minutes),DIEA-CH₂Cl₂ (1:19, 3×2 minutes), CH₂Cl₂ (3×2 minutes),diethyldithiocarbamic acid, sodium salt (0.03 M in DMF, 3×15 minutes),DMF (5×2 minutes), CH₂Cl₂ (3×2 minutes) and DMF (3×1 minute). Fmoc wasremoved with piperidine-DMF (1:4, 3×1 minute+2×5 minutes+2×1 minute) andthe peptide cyclized on resin in CH₂Cl₂ by treatment with PyAOP (5equivalents), HOAt (5 equivalents) and DIEA (10 equivalents) for 2hours, at which time the resin was negative to the Kaiser ninhydrintest. The peptide was cleaved from the resin by treatment withTFA-Et₃SiH-H₂O (92:5:3) at 25° C. for 3 hours and by HPLC showed asingle major component (t_(R) 21.0 minutes; 4.6×250 mm Nucleosil column,linear gradient over 30 minutes of 0.1% TFA in CH₃CN and 0.1% aqueousTFA from 1:9 to 4:6, flow rate 1.0 mL/minute, UV detection at 220 nm,64% purity). MALDI-TOF MS: m/z calcd 1193.6; found 1193.7 [MH³⁰ ].

Example 16 Ethyl 5-(4-formyl-3-hydroxyphenoxy)valerate

Potassium tert-butoxide (2.1 g, mw 112.2, 19 mmol) was added to asolution of 2,4-dihydroxybenzaldehyde (2.5 g, mw 138.1, 18 mmol) in dryDMF (18 mL) and the resultant suspension stirred under N₂ at 25° C. for5 minutes. Ethyl 5-bromovalerate (2.9 mL, mw 209. 1, ρ 1.32, 19 mmol)dissolved in dry DMF (18 mL) was added, and the reaction heated at 110°C. for 5 hours, following which DMF was removed at 60° C. and 2 mm Hg.The residue was taken up in ethyl acetate (100 mL), washed with water(50 mL), and extracted with 1 N aqueous NaOH (50 mL). The ethyl acetatephase was washed with brine (2×50 mL), dried over Na₂SO₄, andconcentrated to yield 1.69 g of an amber colored solid. The NaOH phasewas acidified to pH 3 with 6 N aqueous HCl and extracted with ethylacetate (3×25 mL). The organic extract was washed with brine (2×50 mL),dried over Na₂SO₄, filtered and concentrated to yield 2.54 g of a verydark solid. TLC (CHCl₃-methanol, 99:1) indicated that both solids werecomposed of the same two products (R_(f) 0.37, 0.60), thus they werecombined and purified by flash chromatography on silica (CHCl₃-methanol,99:1) to yield 1.69 g (35%) of title product (R_(f) 0.37) and 0.44 g ofthe dialkylated product, diethyl6-formyl-1,3-phenylenebis(5-oxyvalerate). Both compounds were NMR andTLC pure white solids. For the title product, ¹H NMR (300 MHz, CDCl₃,294 K) δ11.46 (s; OH), 9.70 (s; CHO), 7.41 (d, J=8.6; 1H), 6.51 (dd,J=2.2, 8.6; 1H), 6.39 (d, J=2.2; 1H), 4.13 (q, J=7.1; 2H), 4.02 (t,J=5.7; 2H), 2.37 (t, J=6.9; 2H), 1.82 (m; 4H), 1.25 (t, J=7.1; 3H).

FABMS m/z calcd for C₁₄H₁₈O₅: 266.3, found: 267.1 [MH³⁰ ].

For the dialkylated product, ¹H NMR (300 MHz, CDCl₃, 294 K) δ10.29 (s),7.76 (d, J=8.7; 1H), 6.48 (dd, J=1.9, 8.7; 1H), 6.38 (d, J=1.9; 1H),4.10 (q, J=7.1; 4H), 4.01 (overlapping t; 4H), 2.37 (overlapping t; 4H),1.80 (m; 8H), 1.23 (t, J=7.1; 6H).

FABMS m/z calcd for C₂₁H₃₀O₇: 394.5, found: 395.2 [MH⁺].

Example 17 5-(4-Formyl-3-hydroxyphenoxy)valeric acid

2 N aqueous NaOH (10 mL) was added to ethyl5-(4-formyl-3-hydroxyphenoxy)valerate (1.5 g, mw 266.3, 5.8 mmol;prepared as in Example 16) dissolved in methanol (20 mL) and thesolution stirred at 25° C. for 1 hour, at which point TLC(CHCl₃-methanol, 99:1) indicated the reaction was complete. Methanol wasremoved under reduced pressure and the reaction diluted with water (50mL), washed with ethyl acetate (3×15 mL, discarded), acidified to pH 4with 6 N aqueous HCl, and extracted with ethyl acetate (4×25 mL). Thecombined organic extracts were washed with brine (2×50 mL), dried overNa₂SO₄, filtered, and concentrated to yield 1.35 g (98%) of a whitesolid. A portion of the solid was purified by flash chromatography onsilica (CHCl₃-methanol, 45:1) and recrystallized from boiling ethylacetate-hexane to provide NMR and TLC (R_(f) origin) pure title product;¹H NMR (500 MHz, CDCl₃, 294 K) δ11.47 (s), 9.71 (s), 7.42 (d, J=8.6;1H), 6.53 (dd, J=2.3, 8.6; 1H), 6.41 (d, J=2.3; 1H), 4.04 (t, J=5.8;2H), 2.46 (t, J=7.0; 2H), 1.86 (m; 4H).

Example 18 Ethyl 5-(4-formyl-2,6-dimethylphenoxy)valerate

Potassium tert-butoxide (2.1 g, mw 112.2, 18 mmol) was added to asolution of 4-formyl-2,6-dimethylphenol (2.5 g, mw 150.2, 17 mmol) indry DMF (15 mL) and the resultant suspension stirred under N₂ at 25° C.for 5 minutes. Ethyl 5-bromovalerate (2.8 mL, mw 209.1, ρ 1.32, 18 mmol)dissolved in dry DMF (15 mL) was added, and the reaction heated at 110°C. for 5 hours, following which DMF was removed at 60° C. and 2 mm Hg.The residue was taken up in ethyl acetate (125 mL), washed with water(25 mL), 1 N aqueous NaOH (25 mL), and brine (75 mL), dried over Na₂SO₄,filtered, and concentrated to yield 4.47 g (97%) of NMR and TLC (R_(f)0.58, CHCl₃-methanol, 99:1) pure title product as an amber oil.

¹H NMR (300 MHz, CDCl₃, 294 K) δ9.87 (s), 7.55 (s; 2H), 4.15 (q, J=7.1;2H), 3.83 (t, J=6.0; 2H), 2.41 (t, J=7.0; 2H), 2.33 (s; 6H), 1.87 (m;4H), 1.26 (t, J=17.1; 3H).

Example 19 5-(4-Formyl-2,6-dimethylphenoxy)valeric Acid

2 N aqueous NaOH (30 mL) was added to ethyl5-(4-formyl-2,6-dimethylphenoxy)valerate (4.0 g, mw 278.4, 14 mmol;prepared as in Example 18) dissolved in methanol (40 mL) and that thesolution stirred at 25° C. for 1 hour, at which point TLC(CHCl₃-methanol, 99:1) indicated the reaction was complete. Methanol wasremoved under reduced pressure and the reaction diluted with water (150mL), washed with ethyl acetate (2×25 mL, discarded), acidified to pH 4with 6 N aqueous HCl, and extracted with ethyl acetate (3×30 mL). Theorganic extract was washed with brine (2×75 mL), dried over Na₂SO₄,filtered and concentrated to yield 3.57 g (99%) of an amber oil. Aportion of the oil was purified by flash chromatography on silica(CHCl₃-methanol, 45:1) to provide NMR and TLC (R_(f) origin) pure titleproduct:

¹H NMR (500 MHz, CDCl₃, 294 K) δ9.87 (s; CHO), 7.55 (s; 2H), 3.84 (t,J=5.9; 2H), 2.49 (t, J=7.0; 2H), 2.33 (s; 6H), 1.90 (m; 4H).

FABMS m/z calcd: 250.3 for C₁₄H₁₈O₄, found: 251.1 [MH⁺].

Example 205-(4-(N-Fmoc-N-methyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid

Methylamine (13.3 mL of a 2 M solution in MeOH, 26.6 mmol) was added toa solution of 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid (5.0 g, 17.7mmol) in MeOH (150 mL) under N₂ at 0° C. The reaction was allowed towarm to 25° C., stirred at reflux for 2 hours, and then cooled to 25° C.NaBH₃CN (1.67 g, 26.6 mmol) was added, and the reaction stirred for 1hour at 25° C. Solvent was removed under reduced pressure, and theresidue was taken up in dioxane-10% Na₂CO₃ (1:1, 170 mL) and cooled to0° C. Fmoc-OSu (6.6 g, 19.5 mmol) dissolved in minimal dioxane(approximately 10 mL) was added slowly, and the reaction stirred for 15hours at 25° C. The reaction was washed with ether (3×75 mL), dilutedwith water (150 mL), and carefully acidified to pH 3.0 with 6 N HClunder cooling. The aqueous mixture was extracted with EtOAc (4×50 mL),and the combined organic extracts were washed with brine (2×100 mL),dried (Na₂SO₄), concentrated, and dried in vacuo to yield 6.78 g (74%)of the title product as an off-white solid. The product was crystallizedfrom ether-hexane (65% recovery); mp 126.5-127.5° C.

¹H NMR (300 MHz, CDCl₃, 294μ) δ7.76 (d, J=7.3 Hz, 2H), 7.71 (d, J=7.3Hz, 1H), 7.61 (d, J=7.3 Hz, 1H), 7.39 (dd, J=7.3, 7.3 Hz, 2H), 7.30 (dd,J=7.3, 7.3 Hz, 2H), 6.12 (s, 2H), 4.66 (s, 1H), 4.59 (s, 1H), 4.39 (m,2H), 4.30 (m, 1H), 4.00 (t, J=5.5 Hz, 2H), 3.77 (s, 3H), 3.74 (s, 3H),2.73 (s, 3H), 2.47 (t, J=6.8 Hz, 2H), 1.86 (m, 4H).

FABMS, calcd for C₃₀H₃₃NO₇: 519.6, found: m/z 520.2 [MH⁺].

Elemental analysis: calcd for C₃₀H₃₃NO₇, mw 519.60: C, 69.35; H, 6.40;N, 2.70; found: C, 69.20; H, 6.42; N, 2.72.

Example 21 5-(4-(N-Fmoc-N-ethyl)aminomethyl-3,5-dimethoxyphenoxy)valericacid

The same procedures as in Example 20 were followed, except thatmethylamine was substituted with ethylamine (2 M in MeOH, 18 mmol scale,61% yield); mp 114.8-115.3° C.

¹H NMR (300 MHz, CDCl₃, 294μ) δ7.76 (d, J=7.1 Hz, 2H), 7.71 (d, J=7.1Hz, 1H), 7.60 (d, J=7.1 Hz, 1H), 7.39 (dd, J=7.1, 7.1 Hz, 2H), 7.29 (dd,J=7.1, 7.1 Hz, 2H), 6.11 (s, 2H), 4.67 (s, 1H), 4.57 (s, 1H), 4.41 (m,2H), 4.30 (m, 1H), 3.99 (m, 2H), 3.76 (s, 3H), 3.74 (s, 3H), 3.16 (q,J=6.7Hz, 1H), 3.07 (q, J=6.7 Hz, 1H), 2.47 (m, 2H), 1.86 (m, 4H), 1.01(t, J=6.7, 1.5H), 0.90 (t, J=6.7 Hz, 1.5H).

FABMS, calcd for C₃₁H₃₅NO₇: 533.6, found: m/z 534.3 [MH⁺].

Elemental analysis: calcd for C₃₁H₃₅NO₇, mw 533.63: C, 69.77; H, 6.61;N, 2.63; found: C, 69.69; H, 6.60; N, 2.65.

Example 225-(4-(N-Fmoc-N-phenethyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid

The same procedures as in Example 20 were followed, except thatmethylamine was substituted with phenethylamine (18 mmol scale, 85%yield); mp 141.5-144.5° C.

1H NMR (300 MHz, CDCl₃, 294μ) δ7.8-7.1 (m, 13H), 6.11 (s, 1H), 6.04 (s,1H), 4.66 (s, 1H), 4.57 (s, 1H), 4.50 (d, J=6.7 Hz, 1H), 4.42 (d, J=6.7Hz, 1H), 4.29 (t, J=6.7 Hz, 0.5H), 4.22 (t, J=6.7 Hz, 0.5H), 3.98 (m,2H), 3.74 (s, 6H), 3.29 (t, J=6.7 Hz, 1H), 3.10 (t, J=6.7 Hz, 1H), 2.72(t, J=6.7 Hz, 1H), 2.45 (m, 3H), 1.84 (m, 4H).

FABMS, calcd for C₃₇H₃₉NO₇: 609.7, found: m/z 610.3 [MH⁺].

Elemental analysis: calcd for C₃₇H₃₉NO₇, mw 609.73: C, 72.89; H, 6.45;N, 2.30; found: C, 71.35; H, 6.39; N, 2.59.

Example 235-(4-(N-4-nitrophenyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid

A solution of aldehyde 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid(2.5 g, 8.86 mmol), 4-nitroaniline (1.8 g, 13.3 mmol), and NaBH₃CN (0.83g, 13.3 mmol) was refluxed in MeOH (100 mL) for 15 hours under N₂. Thereaction was allowed to cool to 25° C., and solvent was removed underreduced pressure. The residue was dissolved in 1 N aqueous NaOH (200mL), washed with EtOAc (2×100 mL), and the organic wash wasback-extracted with 1 N aqueous NaOH (100 mL). The combined basicfractions were acidified to pH 4 with 6 N HCl and extracted with EtOAc(4×50 mL). The organic extract was washed with brine (100 mL), dried(Na₂SO₄), concentrated, and dried in vacuo to yield 3.2 g (89%) of crudetitle product as a yellow solid; mp 138.5-139.5° C.; ¹H NMR (CDCl₃)δ8.05 (d, J=9.2, 2H), 6.63 (d, J=9.2 Hz, 2H), 6.12 (s, 2H), 4.37 (s,2H), 3.97 (broad t, 2H), 3.84 (s, 6H), 2.45 (broad t, 2H), 1.84 (m, 4H).

FABMS, calcd for C₂₀H₂₄N₂O₇: 404.4, found: m/z 405.1 [MH⁺].

Elemental analysis: calcd for C₂₀H₂₄N₂O₇, mw 404.42: C, 59.40; H, 5.98;N, 6.93; found: C, 59.20; H, 5.86; N, 6.76.

Example 24 Preparation of Fmoc(R)-PAL-PEG-PS resins

Fmoc-(R)PAL-OH (prepared as in Examples 20-22, 3 equivalents), BOP (3equivalents), HOBt (3 equiv), N-methyl morpholine (6 equivalents), andPEG-PS (0.18 mmol/g, 1 equivalents) were combined in DMF and reacted for15 hours at 25° C., at which time the resins were all negative to theKaiser ninhydrin test; final loadings: 0.16 mmol/g, based on UVquantification of released Fmoc.

Example 25 Synthesis of Des-Gly¹⁰, methylamide⁹-LHRH(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NHMe), (SEQ ID NO: 3)

Fmoc(Me)-PAL-PEG-PS resin (prepared as in Example 24; 150 mg, 0.024mmol) was placed in a manually operated low-pressure continuous-flowsynthesizer with a moving piston. The peptide was assembled using thesame procedure as in Example 10, except that the couplings weremonitored visually using bromophenol blue. Fmoc deprotection wasaccomplished with piperidine-DMF (1:4, 2×10 minutes) and was monitoredbased on UV absorbance. The final peptidyl-resin was washed with DMF andMeOH, and dried in a stream of N₂ (yield: 165 mg). A portion (72 mg) wascleaved and deprotected with TFA-thioanisole-phenol-1,2-ethanedithiol(87:5:5:3, 0.5 mL) for 90 minutes at 25° C. The resin was removed byfiltration and washed with TFA (2×0.5 mL) and CH₂Cl₂ (2×0.5 mL). Thecombined filtrate and washings were concentrated, and the residue washedtwice with diethyl ether. The crude peptide was taken up in 10% aqueousHOAc for final deprotection of Trp, and lyophilized (yield: 14 mg).Amino acid composition of cleaved peptide: Glu, 1.00; His, 0.92; Ser,0.94; Tyr, 0.96; Gly, 1.07; Leu, 1.02; Arg, 1.08; Pro, 1.01. FABMS,calcd: 1139.4, found: m/z 1140.1 [MH⁺].

Example 26 Synthesis of Des-Gly¹⁰, ethylamide⁹-LHRH(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Ar g-Pro-NHEt), (SEQ ID NO: 4)

The same procedures as described in Example 25 were followed except thatFmoc(Me)-PAL-PEG-PS resin was substituted with Fmoc(Et)-PAL-PEG-PS resin(prepared as in Example 24; 500 mg, 0.09 mmol; yield: 580 mg).Peptidyl-resin (270 mg) was then cleaved and deprotected to give thefree peptide (yield: 49 mg). Amino acid composition of cleaved peptide:Glu, 0.97; His, 0.94; Ser, 1.09; Tyr, 0.95; Gly, 1.07; Leu, 0.98; Arg,1.08; Pro, 0.92. FABMS, calcd: 1153.4, found: m/z 1154.5 [MH⁺].

Abbreviations

Abbreviations used for amino acids and the designations of peptidesfollow the rules of the IUPAC-IUB Commission of Biochemical Nomenclaturein J. Biol. Chem. 1972, 247, 977-983. The following additionalabbreviations are used: Ac, acetyl; Al, allyl; Boc,tert.-butyloxycarbonyl; BOP,benzotriazolyl-N-oxytris(dimethylamino)phosphonium hexafluorophosphate;DIEA, N,N-diisopropylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide;DMF, N,N-dimethylformamide; ESMS, electrospray mass spectrometry;Fmoc-OSu, N-(9-fluorenylmethoxycarbonyloxy)succinimide; Fmoc-Cl,9-fluorenylmethyl chloroformate; HATU,N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; HPLC,high-performance liquid chromatography; HOBt, 1-hydroxybenzotriazole;FABMS, fast atom bombardment mass spectrometry; Fmoc,9-fluorenylmethoxycarbonyl; MALDI-TOF, matrix assisted laserdesorption/ionization time-of-flight mass spectrometry; MBHA,methylbenzhydrylamine (resin); PEG-PS, polyethylene glycol-polystyrenegraft support; Pfp, pentafluorophenyl; PyAOP,7-azabenzotriazol-1-yl-oxytris(pyrrolidino)phosphoniumhexafluorophosphate; SPPS, solid-phase peptide synthesis; PALdehyde,5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid; o,p-PALdehyde, mixture of5-(4-formyl-3,5-dimethoxyphenoxy)butyric acid and5-(2-formyl-3,5-dimethoxyphenoxy)butyric acid; TFA, trifluoroaceticacid; Trt, trityl. Aloc, allyloxycarbonyl; Ddz,2-(3,5-dimethoxyphenyl)isopropoxycarbonyl; Npys,3-nitro-2-pyridinesulfenyl; Nvoc, 6-nitroveratryloxycarbonyl; Bpoc,biphenylisopropoxycarbonyl; Teoc, 2-(trimethylsilyl)ethoxycarbonyl; SES,β-trimethylsilylethane sulfonyl; t-Bu, tert-butyl; Pmc,2,2,5,7,8-pentamethylchroman-6-sulfonyl; HOAt,3-hydroxy-3H-1,2,3-triazolo[4,5,b]pyridine. Amino acid symbols denotethe L-configuration unless stated otherwise. All solvent ratios arevolume/volume unless stated otherwise.

The complete disclosure of all patents, patent documents, andpublications cited herein are incorporated by reference. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

4 1 7 PRT Artificial Sequence Description of Artificial Sequencemodelpeptide aldehyde 1 Ala Leu Ala Lys Leu Gly Xaa 1 5 2 6 PRT ArtificialSequence Description of Artificial Sequencemodel peptide 2 Tyr Gly GlyPhe Xaa Xaa 1 5 3 9 PRT Artificial Sequence Description of ArtificialSequenceDes-Gly10, methylamide9-LHRH 3 Xaa His Trp Ser Tyr Gly Leu ArgXaa 1 5 4 9 PRT Artificial Sequence Description of ArtificialSequenceDes-Gly10, ethylamide9-LHRH 4 Xaa His Trp Ser Tyr Gly Leu ArgXaa 1 5

What is claimed is:
 1. An aldehyde-functionalized support materialhaving the following formula (Formula III):

wherein: (a) Ŝ represents a support material; (b) V is NH, S or O; (c)R⁷ is an alkylene group, an arylene group, or an oxyalkylene group; (d)T is O, NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group, an arylenegroup, or an aralkylene group; (e) each U is independently selected fromthe group consisting of an alkyl group, an alkoxy group, an aryl group,an alkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthiogroup, an arylthio group, an alkylamido group, an alkylsulfinyl group, ahalogeno group, and a nitro group, wherein any two U groups call bejoined to form a ring; (f) x=0-4; and (g) n=1-18, with the proviso thatthe compound with V=NH, R⁷=methylene, n=1, T=oxygen, x=0 and thealdehyde group para to T is excluded.
 2. The aldehyde-functionalizedsupport material of claim 1 wherein R⁷ is an alkylene group and n=1-4.3. A kit for synthesizing an organic compound comprising: (a) analdehyde-functionalized support material having the following formula(Formula III):

 wherein: (i) Ŝ represents a support material; (ii) V is NH, S or O;(iii) R⁷ is an alkylene group, an arylene group, or an oxyalkylenegroup; (iv) T is O, NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group,an arylene group, or an aralkylene group; (v) each U is independentlyselected from the group consisting of an alkyl group, an alkoxy group,an aryl group, an alkoxyaryl group, an aralkyl group, an aralkoxy group,an alkylthio group, an arylthio group, an alkylamido group, analkylsulfinyl group, a halogeno group, and a nitro group, wherein anytwo U groups can be joined to form a ring; (vi) x=0-4; and (vii) n=1-18,with the proviso that the compound with V=NH, R⁷=methylene, n=1,T=oxygen, x=0, and the aldehyde group para to T is excluded; and (b)instructions for preparing an organic compound on thealdehyde-functionalized support material.
 4. An aldehyde-functionalizedsolid support material having the following formula (Formula III):

wherein: (i) Ŝ represents a solid support material; (ii) R⁷ is analkylene group, an arylene group, or an oxyalkylene group; (ii) T is O,NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group, an arylene group,or an aralkylene group; (iv) each U is independently selected from thegroup consisting of an alkyl group, an alkoxy group, an aryl group, analkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthiogroup, an arylthio group, an alkylamido group, an alkylsulfinyl group, ahalogeno group, and a nitro group, wherein any two U groups can bejoined to form a ring; (v) x=0-4, and (vi) n=1-18, with the proviso thatthe compound with R⁷=methylene, n=1, T=oxygen, x=0, and the aldehydegroup para to T is excluded.
 5. The aldehyde-functionalized supportmaterial of claim 4 wherein R⁷ is an alkylene group and n=1-4.
 6. A kitfor synthesizing an organic compound comprising: (a) an)aldehyde-functionalized solid support material having the followingformula (Formula III):

 wherein: (i) Ŝ represents a solid support material; (ii) R⁷ is analkylene group, an arylene group, or an oxyalkylene group; (iii) T is O,NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group, an arylene group,or an aralkylene group; (iv) each U is independently selected from thegroup consisting of an alkyl group, an alkoxy group, an aryl group, analkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthiogroup, an arylthio group, an alkylamido group, an alkylsulfinyl group, ahalogeno group, and a nitro group, wherein any two U groups can bejoined to form a ring; (v) x=0-4; and (vi) n=1-18, with the proviso thatthe compound with R⁷=methylene, n=1, T=oxygen, x=0, and the aldehydegroup para to T is excluded; and (b) instructions for preparing anorganic compound on the aldehyde-functionalized support material.
 7. Analdehyde-functionalized support material having the following formula(Formula III):

wherein: (a) Ŝ represents a support material; (b) V is S or O; (c) R⁷ isan alkylene group, an arylene group, or an oxyalkylene group; (d) T isO, NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group, an arylenegroup, or an aralkylene group; (e) each U is independently selected fromthe group consisting of an alkyl group, an alkoxy group, an aryl group,an alkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthiogroup, an arylthio group, an alkylamido group, an alkylsulfinyl group, ahalogeno group, and a nitro group, wherein any two U groups can bejoined to form a ring; (f) x=0-4; and (g) n=1-18.
 8. Thealdehyde-functionalized support material of claim 7 wherein R⁷ is analkylene group and n=1-4.
 9. An aldehyde-functionalized support materialhaving the following formula (Formula III):

wherein: (a) Ŝ represents a support material; (b) V is NH, S or O; (c)R⁷ is an alkylene group, an arylene group, or an oxyalkylene group; (d)T is O, NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group, an arylenegroup, or an aralkylene group; (e) each U is independently selected fromthe group consisting of an alkyl group, an alkoxy group, an aryl group,an alkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthiogroup, an arylthio group, an alkylamido group, an alkylsulfinyl group, ahalogeno group, and a nitro group, wherein any two U groups can bejoined to form a ring; (f) x=1-4; and (g) n=1-18.
 10. Thealdehyde-functionalized support material of claim 9 wherein R⁷ is analkylene group and n=1-4.
 11. A kit for synthesizing an organic compoundcomprising: (a) an aldehyde-functionalized support material having thefollowing formula (Formula III):

 wherein: (i) Ŝ represents a support material; (ii) V is NH, S or O;(iii) R⁷ is an alkylene group, an arylene group, or an oxyalkylenegroup; (iv) T is O, NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group,an arylene group, or an aralkylene group; (v) each U is independentlyselected from the group consisting of an alkyl group, an alkoxy group,an aryl group, an alkoxyaryl group, an aralkyl group, an aralkoxy group,an alkylthio group, an arylthio group, an alkylamido group, analkylsulfinyl group, a halogeno group, and a nitro group, wherein anytwo U groups can be joined to form a ring; (vi) x=1-4, and (vii) n=1-18;and (b) instructions for preparing an organic compound on thealdehyde-functionalized support material.
 12. An aldehyde-functionalizedsolid support material having the following formula (Formula III):

wherein: (i) Ŝ represents a solid support material; (ii) R⁷ is analkylene group, an arylene group, or an oxyalkylene group; (iii) T is O,NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group, an arylene group,or an aralkylene group; (iv) each U is independently selected from thegroup consisting of an alkyl group, an alkoxy group, an aryl group, analkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthiogroup, an arylthio group, an alkylamido group, an alkylsulfinyl group, ahalogeno group, and a nitro group, wherein any two U groups can bejoined to form a ring; (v) x=1-4; and (vi) n=1-18.
 13. Thealdehyde-functionalized support material of claim 12 wherein R⁷ is analkylene group and n=1-4.
 14. A kit for synthesizing an organic compoundcomprising: (a) an aldehyde-functionalized solid support material havingthe following formula (Formula III):

 wherein: (i) Ŝ represents a solid support material; (ii) R⁷ is analkylene group, an arylene group, or an oxyalkylene group; (iii) T is O,NH, NHC(O)R⁴, or S, wherein R⁴ is an alkylene group, an arylene group,or an aralkylene group; (iv) each U is independently selected from thegroup consisting of an alkyl group, an alkoxy group, an aryl group, analkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthiogroup, an arylthio group, an alkylamido group, an alkylsulfinyl group, ahalogeno group, and a nitro group, wherein any two U groups can bejoined to form a ring; (v) x=1-4; and (vi) n=1-18; and (b) instructionsfor preparing an organic compound on the aldehyde-functionalized supportmaterial.